In most cells MHC class I molecules exclusively display peptides derived from the cell's own proteins. However, dendritic cells and macrophages have the unique capability to capture and present antigens from the environment on MHC class I molecules through a process called cross presentation. This pathway is important because it plays an essential role in the immune surveillance of tissues for viral infections and cancers and chronically infected phagocytes. It is also a potential portal for vaccine delivery. The overall goal of this proposal is to elucidate the mechanisms that underlie cross presentation. We have previously defined two major intracellular mechanisms - the vacuolar and phagosome-to-cytosol (P2C) pathways - through which exogenous proteins are cross presented both in vitro and in vivo. Our first Aim will address the mystery as to why some antigens are cross-presented exclusively by the P2C mechanism while others are also presented through the vacuolar pathway. Our underlying hypothesis is that these two pathways operate in distinct vesicular compartments that are accessed by different exogenous antigens. Our second Aim will address the major unresolved issue of how antigens in the P2C pathway are dislocated from phagosomes into the cytosol. We hypothesize that two distinct mechanisms may be involved. The first is the transport of antigen through an ERAD-like pathway. The second is a rupture of the phagosomal membrane;we further hypothesize that this rupture may be facilitated by oxidative damage to the phagosomal membrane. Our third Aim will define how and where MHC class I molecules sample exogenous cross-presented peptides in the vacuolar and P2C pathways. PUBLIC HEALTH RELEVANCE: Narrative. This proposal seeks to elucidate how the immune system monitors tissues virally infected cells and cancers. This is an important issue because this process is essential to our ability to eliminate or prevent these pathological processes. The information gained by the proposed studies may lead to a better ability to predict and monitor immune responses to viruses and cancers and ultimately aid in the development of vaccines for these diseases.